JP3407302B2 - Method for identifying a microorganism using at least two types of chromogens - Google Patents
Method for identifying a microorganism using at least two types of chromogensInfo
- Publication number
- JP3407302B2 JP3407302B2 JP50562495A JP50562495A JP3407302B2 JP 3407302 B2 JP3407302 B2 JP 3407302B2 JP 50562495 A JP50562495 A JP 50562495A JP 50562495 A JP50562495 A JP 50562495A JP 3407302 B2 JP3407302 B2 JP 3407302B2
- Authority
- JP
- Japan
- Prior art keywords
- medium
- chromogen
- indoxyl
- color
- chromogens
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 238000000034 method Methods 0.000 title claims abstract description 27
- 244000005700 microbiome Species 0.000 title claims abstract description 18
- 239000000758 substrate Substances 0.000 claims abstract description 7
- 102000004190 Enzymes Human genes 0.000 claims abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 6
- 239000002609 medium Substances 0.000 claims description 21
- PCKPVGOLPKLUHR-UHFFFAOYSA-N indoxyl Chemical group C1=CC=C2C(O)=CNC2=C1 PCKPVGOLPKLUHR-UHFFFAOYSA-N 0.000 claims description 14
- 150000001875 compounds Chemical class 0.000 claims description 12
- 239000003086 colorant Substances 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 150000001720 carbohydrates Chemical class 0.000 claims description 5
- 235000014633 carbohydrates Nutrition 0.000 claims description 5
- 241000588914 Enterobacter Species 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- 230000000813 microbial effect Effects 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 3
- 241000588923 Citrobacter Species 0.000 claims description 3
- 241000588724 Escherichia coli Species 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 241000588748 Klebsiella Species 0.000 claims description 3
- 241000194017 Streptococcus Species 0.000 claims description 3
- 238000005859 coupling reaction Methods 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 230000007062 hydrolysis Effects 0.000 claims description 3
- 238000006460 hydrolysis reaction Methods 0.000 claims description 3
- AXCKOXONHIRKQP-UHFFFAOYSA-N 5-bromo-4-chloro-3-hydroxyindole Chemical group C1=C(Br)C(Cl)=C2C(O)=CNC2=C1 AXCKOXONHIRKQP-UHFFFAOYSA-N 0.000 claims description 2
- 241000193403 Clostridium Species 0.000 claims description 2
- 241000588722 Escherichia Species 0.000 claims description 2
- 241000186781 Listeria Species 0.000 claims description 2
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- 241000588769 Proteus <enterobacteria> Species 0.000 claims description 2
- 241000191940 Staphylococcus Species 0.000 claims description 2
- 239000007640 basal medium Substances 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims 1
- 239000001963 growth medium Substances 0.000 abstract 3
- 102220201851 rs143406017 Human genes 0.000 description 4
- 241000222122 Candida albicans Species 0.000 description 3
- 229940095731 candida albicans Drugs 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 241000233866 Fungi Species 0.000 description 2
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 2
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 2
- 239000003593 chromogenic compound Substances 0.000 description 2
- 238000004040 coloring Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- LBXRGISCDMNDSD-UHFFFAOYSA-N 5-bromo-3-hydroxyindole Chemical group C1=C(Br)C=C2C(O)=CNC2=C1 LBXRGISCDMNDSD-UHFFFAOYSA-N 0.000 description 1
- 241000222178 Candida tropicalis Species 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical class N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 102000005262 Sulfatase Human genes 0.000 description 1
- 102000005840 alpha-Galactosidase Human genes 0.000 description 1
- 108010030291 alpha-Galactosidase Proteins 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 108010012864 alpha-Mannosidase Proteins 0.000 description 1
- 102000019199 alpha-Mannosidase Human genes 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 150000005209 naphthoic acids Chemical class 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 108060007951 sulfatase Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/10—Enterobacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/50—Indoles
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/50—Indoles
- C12Q2334/52—5-Bromo-4-chloro-3-indolyl, i.e. BCI
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
本発明は、培地中に特定の微生物菌株が存在するかど
うかを検出するための方法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for detecting the presence of a particular microbial strain in a medium.
微生物が病原性であるかもしれないだけでなく、何ら
かの種類の汚染を示す可能性があることから、微生物の
検出は非常に重要であり、特に食品工業、水質管理、ま
たは医薬において重要である。The detection of microorganisms is of great importance, especially in the food industry, water quality management, or medicine, as the microorganisms may not only be pathogenic but may exhibit some kind of contamination.
ある種の培地中において微生物の存在を検出すること
ができる方法は種々あり、それらは、対象とする培地の
試料を採取し、次いで適宜培地上または培地中で培養す
ることにより存在する微生物の成長を促進することから
なる。There are various methods by which the presence of microorganisms can be detected in certain media, which involves the growth of the microorganisms present by taking a sample of the medium of interest and then culturing on or in the medium as appropriate. Consists of promoting.
存在する微生物の検出を簡便にするために、検出培地
において、ある微生物に特有の着色化合物を使用するこ
とが提案されてきた。In order to simplify the detection of existing microorganisms, it has been proposed to use a coloring compound specific to a certain microorganism in the detection medium.
発色が、問題の微生物と関連した酵素活性を表すこと
はよくあり、この活性の発現により、着色指示薬により
示される培地のpH変化がもたらされる（EP−Ａ−0 395
532）か、あるいは発色団化合物（chromophoric compou
nd）もしくは発蛍光団化合物（fluorophoric compoun
d）の遊離がもたらされる（FR−Ａ−２−684,110号）。Color development often represents an enzymatic activity associated with the microorganism in question, and expression of this activity results in a pH change in the medium as indicated by the color indicator (EP-A-0 395).
532) or a chromophore compound (chromophoric compou
nd) or fluorophore compoun
This results in the liberation of d) (FR-A-2-684,110).
発色団あるいは発蛍光団とは、一般的には、培地中
の、対応する色原体化合物（発色団を含む化合物、chro
mogenic compound）もしくは発蛍光団を含む化合物（fl
uorogenic compound）が酵素により加水分解されること
により得られる化合物である。A chromophore or fluorophore generally means the corresponding chromogenic compound (compound containing a chromophore, chro
mogenic compound) or a compound containing a fluorophore (fl
uorogenic compound) is a compound obtained by hydrolysis with an enzyme.
発蛍光団は蛍光により特有の光を放射する。 A fluorophore emits a unique light due to fluorescence.
発色団化合物は主波長による色を特徴とする。 Chromophore compounds are characterized by a dominant wavelength color.
既知の発色団化合物としては、特に、インドキシル誘
導体、ヒドロキノリン誘導体、あるいはナフトエ酸誘導
体やナフチルおよびフェニル誘導体が挙げられる。Known chromophore compounds include indoxyl derivatives, hydroquinoline derivatives, or naphthoic acid derivatives and naphthyl and phenyl derivatives, among others.
培地中の異なる２種の属の微生物を区別するために、
それぞれが個々の微生物の存在に特有の色を有する発色
団化合物を遊離するような、２種の色原体を導入する提
案がなされた（US−Ａ−５−210,022）。In order to distinguish between the microorganisms of two different genera in the medium,
It has been proposed (US-A-5-210,022) to introduce two chromogens, each liberating a chromophore compound having a color characteristic of the presence of an individual microorganism.
これらの培地はすべて、特定属の微生物、例えばサル
モネラ、カンジダ、または大腸菌（E.coli）などを検出
し、それらを他の種類と区別するのに有効であるが、同
じ培地上の異なる属の多数の微生物を検出することはで
きず、また同じ属の微生物の中で病原性の種類とその他
を区別できない。All of these media are effective in detecting a specific genus of microorganisms, such as Salmonella, Candida, or E. coli, and distinguishing them from other species, but of different genera on the same media. It is not possible to detect a large number of microorganisms and to distinguish between pathogenic species and others among microorganisms of the same genus.
このような区別はすべて、ある種の酵母、例えば酵母
に関連した病状の50％以上の原因であるカンジダ・アル
ビカンス（Candida albicans）、にとってはより重要で
あるようだ。All such distinctions appear to be more important for certain yeasts, such as Candida albicans, responsible for over 50% of yeast-related pathologies.
ここに、特定の菌株の酵素に対する基質である、次の
ような少なくとも２種の色原体を培地中に導入すること
により、菌株を特徴づける少なくとも４種の異なる発色
がみられることが予想外にも見出された：この２種の色
原体は、培地中において少なくとも１種の菌株の存在が
以下のような第３の色（tierce couleur）（付随色、ac
cessorry color）により明らかになるように選択された
ものである：
−選択した色原体に対応する発色団の２種類の色、
−発色団の混合に対応する色、および
−第３の色（付随色）。It is unexpected that at least four different colorings that characterize the strain are observed by introducing into the medium at least two chromogens, which are substrates for enzymes of a specific strain, as follows. Were also found in: The two chromogens have a third tierce couleur (associated color, ac
The two colors of the chromophores corresponding to the selected chromogen, the color corresponding to the mixture of chromophores, and the third color ( Incidental color).
多くの場合、付随色は、対応する２種の発色団の色の
混合には対応せず、その主波長が、培地中に存在する色
原体から遊離させた別々の発色団の混合物の主波長には
対応しない色である。In many cases, ancillary colors do not correspond to a mixture of the colors of the corresponding two chromophores, the dominant wavelength of which is the major constituent of a mixture of separate chromophores released from the chromogen present in the medium. It is a color that does not correspond to the wavelength.
発色団および付随色の主波長は、対象物の色を測定す
る任意の標準的方法、特に分光比色計を用いて、光源D
65としてCIE（エネルギーに関する国際委員会、Interna
tional Commission on Energy）に定義するように、日
光を対照として評価すればよい。The dominant wavelengths of the chromophore and associated colors can be determined using any standard method of measuring the color of an object, in particular a spectrocolorimeter, with a light source D
65 as CIE (International Commission on Energy, Interna
Sunlight can be assessed as a control, as defined by the National Commission on Energy).
従って、本発明は、培地中の特定の微生物菌株の有無
を検出する方法であり、この特定の微生物菌株の酵素基
質である少なくとも２種の色原体を導入することからな
り、この色原体は培地中において前記菌株の存在が第３
の色（付随色）により現れるように選択されることを特
徴とする。Therefore, the present invention is a method for detecting the presence or absence of a specific microbial strain in a medium, which comprises introducing at least two chromogens that are enzyme substrates of this specific microbial strain. The presence of said strain in the medium is
Is selected to appear according to the color (subordinate color) of.
特に、色原体は次のような酵素の基質である：β−ガ
ラクトシダーゼ、β−グルコシダーゼ、β−グルクロニ
ダーゼ、α−グルコシダーゼ、α−ガラクトシダーゼ、
ホスファターゼ、Ｎ−アセチル−β−グルコシダーゼ、
Ｎ−アセチル−β−ガラクトシダーゼ、α−マンノシダ
ーゼ、スルファターゼ、エステラーゼ、リパーゼおよび
ペプチダーゼ。In particular, chromogens are substrates for the following enzymes: β-galactosidase, β-glucosidase, β-glucuronidase, α-glucosidase, α-galactosidase,
Phosphatase, N-acetyl-β-glucosidase,
N-acetyl-β-galactosidase, α-mannosidase, sulfatase, esterase, lipase and peptidase.
色原体は、化学的に同族の化合物、好ましくはカップ
リング反応を受けることができる２種の異なる発色団を
加水分解により遊離する化合物から有利に選択される。Chromogens are advantageously selected from chemically homologous compounds, preferably compounds which liberate by hydrolysis two different chromophores which can undergo a coupling reaction.
カップリング反応は、得られる主波長が、別々にとっ
た、２種の発色団の混合物の主波長とは異なるように物
理化学的相互作用を意味する。Coupling reaction refers to a physicochemical interaction such that the dominant wavelength obtained is different from the dominant wavelength of a mixture of two chromophores taken separately.
好ましくは、色原体はインドキシル系、殊にアルキル
化、ハロゲン化またはジハロゲン化インドキシル誘導体
である。Preferably, the chromogen is an indoxyl system, especially an alkylated, halogenated or dihalogenated indoxyl derivative.
インドキシルから誘導される好ましい発色団には、ブ
ロモインドキシル、クロロインドキシル、ジクロロイン
ドキシル、クロロブロモインドキシル、トリクロロイン
ドキシルおよびメチルインドキシルのインドキシル誘導
体、殊に次のような誘導体がある:6−クロロインドキシ
ル、５−ブロモインドキシル、３−ブロモインドキシ
ル、4,6−ジクロロインドキシル、6,7−ジクロロインド
キシル、５−ブロモ−４−クロロインドキシル、５−ブ
ロモ−６−クロロインドキシルまたは4,6,7−トリクロ
ロインドキシル。Preferred chromophores derived from indoxyl include bromoindoxyl, chloroindoxyl, dichloroindoxyl, chlorobromoindoxyl, trichloroindoxyl and methylindoxyl indoxyl derivatives, especially the following derivatives: : 6-chloroindoxyl, 5-bromoindoxyl, 3-bromoindoxyl, 4,6-dichloroindoxyl, 6,7-dichloroindoxyl, 5-bromo-4-chloroindoxyl, 5-bromo-6 -Chloroindoxyl or 4,6,7-trichloroindoxyl.
本発明方法によって検出される微生物とは、酵母類、
カビ類または単細胞菌類および細菌類を意味する。Microorganisms detected by the method of the present invention, yeasts,
By fungi or unicellular fungi and bacteria.
本発明方法はカンジダ属、殊にカンジダ・アルビカン
スやカンジダ・トロピカリス（Candida tropicalis）の
酵母類の存在の有無を検出するのに特に適している。The method according to the invention is particularly suitable for detecting the presence or absence of yeasts of the genus Candida, in particular Candida albicans and Candida tropicalis.
同様に、本発明方法によって存在の有無を検出しうる
細菌類には、特にストレプトコッカス（Streptococcu
s）属、クレブシェラ（Klebsiella）属、エンテロバク
ター（Enterobacter）属、エシェリヒア（Escherichi
a）属、シトロバクター（Citrobacter）属、スタフィロ
コッカス（Staphylococcus）属、リステリア（Listeri
a）属、クロストリジウム（Clostridium）属またはプロ
テウス（Proteus）属の細菌類が含まれる。Similarly, bacteria whose presence can be detected by the method of the present invention include Streptococcus, in particular.
s) genus, Klebsiella genus, Enterobacter genus, Escherichia
a) Genus, Citrobacter genus, Staphylococcus genus, Listeria
a) genus, Clostridium genus or Proteus genus bacteria are included.
さらに、培地中に高濃度で炭水化物を添加すると、本
発明方法によって得られる色の数を増加させる。すなわ
ち、限られた数の色原体で、より多くの異なる微生物が
区別できるような多数の異なる色を得ることができるこ
とも見出された。Moreover, the addition of high concentrations of carbohydrates in the medium increases the number of colors obtained by the method of the invention. That is, it was also found that with a limited number of chromogens it is possible to obtain a large number of different colors that allow more different microorganisms to be distinguished.
従って、本発明は、培地が高濃度の炭水化物、好まし
くはグルコースを、ペプトン基本培地中に含有する、上
記方法にも関する。The invention therefore also relates to the above method, wherein the medium contains a high concentration of carbohydrates, preferably glucose, in the peptone basal medium.
炭水化物の高濃度とは、有利には10〜30g/lの範囲で
ある。A high concentration of carbohydrates is advantageously in the range 10-30 g / l.
同様に、色原体のうちの少なくとも１種がホスファタ
ーゼの基質である場合は、高濃度、好ましくは１〜3g/l
の範囲内のホスフェートを含有させるのが有利である。Similarly, when at least one of the chromogens is a substrate for phosphatase, a high concentration, preferably 1-3 g / l
It is advantageous to include a phosphate in the range of
以下に示す例は、本発明方法を説明するものである
が、その範囲を限定するものではない。The following examples illustrate the method of the invention, but do not limit its scope.
標準培地で現れる色は以下の通りである：
ストレプトコッカスＤ（青）、クレブシェラ（青）、エ
ンテロバクター（青）、エンテロバクターMUG＋
（青）、E.coli（無色）シトロバクター（無色）
上記結果は、グルコースおよびホスフェートの添加に
より、同じ培地で利用しうる色の範囲が広がり、この例
では異なる７つの種を区別すること、特にカンジダ・ア
ルビカンスを明確に同定しうることが明らかである。 The colors appearing in the standard medium are as follows: Streptococcus D (blue), Klebsiella (blue), Enterobacter (blue), Enterobacter MUG +.
(Blue), E.coli (colorless) Citrobacter (colorless) The above results show that the addition of glucose and phosphate expands the range of colors available in the same medium and in this example distinguishes seven different species, in particular Candida albicans can be clearly identified. .
フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ (Ｃ１２Ｑ 1/04 Ｃ１２Ｒ 1:01 Ｃ１２Ｒ 1:22） (Ｃ１２Ｑ 1/04 Ｃ１２Ｒ 1:19） (Ｃ１２Ｑ 1/04 Ｃ１２Ｒ 1:01） Front page continuation (51) Int.Cl. 7 Identification code FI (C12Q 1/04 C12R 1:01 C12R 1:22) (C12Q 1/04 C12R 1:19) (C12Q 1/04 C12R 1:01)
Claims (15)
る方法であり、該菌株に対する酵素基質である少なくと
も２種の色原体を培地中に添加し、該色原体は、培地中
における前記菌株の存在が第３の色（付随色）により明
らかにされるように選択され、ここで第３の色とは、前
記色原体に対応する発色団の色の混合には相当せず、そ
してその主波長が、別々に培地中に存在する色原体から
遊離させた発色団の混合物の主波長には対応しないよう
な色であることを特徴とする、前記方法。1. A method for detecting the presence of a specific microbial strain in a medium, wherein at least two chromogens that are enzyme substrates for the strain are added to the medium, and the chromogen is in the medium. The presence of said strain in E. coli was selected to be revealed by a third color (subordinate color), where the third color corresponds to a mixture of chromophore colors corresponding to said chromogen. And a color whose dominant wavelength does not correspond to the dominant wavelength of the mixture of chromophores liberated from the chromogen separately present in the medium.
求の範囲第１項記載の方法。2. The method of claim 1 wherein the chromogen is a chemically homologous compound.
うる、２種の異なる発色団を加水分解により遊離する、
請求の範囲第１項または第２項記載の方法。3. Two chromogens liberate by hydrolysis two different chromophores capable of undergoing a coupling reaction,
The method according to claim 1 or 2.
化、ハロゲン化またはジハロゲン化インドキシル誘導体
である、請求の範囲第１項〜第３項のいずれかに記載の
方法。4. A process according to any one of claims 1 to 3, wherein the chromogen is an indoxyl-based, in particular an alkylated, halogenated or dihalogenated indoxyl derivative.
モインドキシル、クロロインドキシル、ジクロロインド
キシル、クロロブロモインドキシル、トリクロロインド
キシルおよびメチルインドキシルから、特に６−クロロ
インドキシル、５−ブロモインドキシル、３−ブロモイ
ンドキシル、4,6−ジクロロインドキシル、6,7−ジクロ
ロインドキシル、５−ブロモ−４−クロロインドキシ
ル、５−ブロモ−６−クロロインドキシルまたは4,6,7
−トリクロロインドキシルのインドキシル誘導体から選
択される、請求の範囲第１項〜第４項のいずれかに記載
の方法。5. The chromophore is selected from the indoxyl derivatives bromoindoxyl, chloroindoxyl, dichloroindoxyl, chlorobromoindoxyl, trichloroindoxyl and methylindoxyl, especially 6-chloroindoxyl, 5-bromo. Indoxyl, 3-bromoindoxyl, 4,6-dichloroindoxyl, 6,7-dichloroindoxyl, 5-bromo-4-chloroindoxyl, 5-bromo-6-chloroindoxyl or 4,6,7
-The method according to any of claims 1 to 4, selected from an indoxyl derivative of trichloroindoxyl.
〜第５項のいずれかに記載の方法。6. The method according to any one of claims 1 to 5, wherein the microorganism is yeast.
求の範囲第６項に記載の方法。7. The method according to claim 6, wherein the yeast belongs to the genus Candida.
〜第５項のいずれかに記載の方法。8. The method according to any one of claims 1 to 5, wherein the microorganism is a bacterium.
エラ属、エンテロバクター属、エシェリヒア属、シトロ
バクター属、スタフィロコッカス属、リステリア属、ク
ロストリジウム属またはプロテウス属に属するものであ
る、請求の範囲第８項に記載の方法。9. The method according to claim 8, wherein the bacterium belongs to the genus Streptococcus, Klebsiella, Enterobacter, Escherichia, Citrobacter, Staphylococcus, Listeria, Clostridium or Proteus. The method described in.
請求の範囲第１項〜第９項のいずれかに記載の方法。10. The medium contains a high concentration of carbohydrates,
The method according to any one of claims 1 to 9.
ルコースを含有するものである、請求の範囲第10項に記
載の方法。11. The method according to claim 10, wherein the medium is a peptone basal medium containing a high concentration of glucose.
ある、請求の範囲第10項および第11項のいずれかに記載
の方法。12. A method according to any of claims 10 and 11 wherein the concentration of carbohydrate is in the range 10-30 g / l.
ある、請求の範囲第１項〜第12項のいずれかに記載の方
法。13. The method according to any one of claims 1 to 12, wherein one chromogen is a substrate for phosphatase.
る、請求の範囲第13項に記載の方法。14. The method according to claim 13, wherein the medium contains a high concentration of phosphate.
である、請求の範囲第14項に記載の方法。15. The method according to claim 14, wherein the concentration of phosphate is in the range of 1 to 3 g / l.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9309294A FR2708286B1 (en) | 1993-07-28 | 1993-07-28 | Method of identifying microorganisms with at least two chromogens. |
| FR93/09294 | 1993-07-28 | ||
| PCT/FR1994/000958 WO1995004157A1 (en) | 1993-07-28 | 1994-07-28 | Microorganism identification method using at least two chromogens |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH09500791A JPH09500791A (en) | 1997-01-28 |
| JP3407302B2 true JP3407302B2 (en) | 2003-05-19 |
Family
ID=9449718
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP50562495A Expired - Lifetime JP3407302B2 (en) | 1993-07-28 | 1994-07-28 | Method for identifying a microorganism using at least two types of chromogens |
Country Status (10)
| Country | Link |
|---|---|
| US (1) | US5962251A (en) |
| EP (1) | EP0711360B1 (en) |
| JP (1) | JP3407302B2 (en) |
| AT (1) | ATE163972T1 (en) |
| CA (1) | CA2168114C (en) |
| DE (1) | DE69408993T2 (en) |
| DK (1) | DK0711360T3 (en) |
| ES (1) | ES2114697T3 (en) |
| FR (1) | FR2708286B1 (en) |
| WO (1) | WO1995004157A1 (en) |
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| FR2747394B1 (en) * | 1996-04-15 | 1998-07-03 | Rambach Alain | CULTURE MEDIUM FOR THE EVIDENCE OF ENTEROHEMORRAGIC E. BACTERIA BACTERIA, AND METHOD FOR ITS EVIDENCE |
| ATE226630T1 (en) | 1996-07-26 | 2002-11-15 | Idexx Lab Inc | METHOD AND MEDIUM FOR DETECTING VANCOMYCIN-RESISTANT ENTEROCOCCUS |
| EP0961834B1 (en) | 1997-06-04 | 2003-05-07 | The Newcastle upon Tyne Hospitals National Health Service Trust | Identification of salmonella |
| FR2767535B1 (en) * | 1997-08-20 | 2000-02-04 | Bio Merieux | CULTURE AND SPECIFIC IDENTIFICATION MEDIA FOR DIFFERENT SPECIES OF CANDIDA AND METHODS OF ANALYSIS |
| FR2774097B1 (en) * | 1998-01-28 | 2000-09-29 | Bio Merieux | USE OF INDOLAMINE DERIVATIVES IN THE DETECTION OF PEPTIDASE ACTIVITY OR IN THE DETECTION OF MICROORGANISMS |
| FR2777018B1 (en) | 1998-04-02 | 2003-04-25 | Bio Merieux | NEW CHROMOGENEOUS SUBSTRATE AND METHOD OF USING THE SAME |
| FR2790765B1 (en) | 1999-03-11 | 2003-01-31 | Alain Rambach | CHROMOGENEOUS ENVIRONMENT FOR THE DETECTION OF STAPHYLOCOCCUS AUREUS. |
| US7344854B2 (en) * | 1999-07-20 | 2008-03-18 | Micrology Laboratories, Llc | Test media for quantitative or qualitative identification and differentiation of general coliforms, E. coli, Aeromonas spp and Salmonella spp in a test sample |
| US6350588B1 (en) | 1999-07-20 | 2002-02-26 | Micrology Laboratories, Llc | Test media and quantitative or qualitative method for identification and differentiation of biological materials in a test sample |
| US7273719B2 (en) * | 1999-07-20 | 2007-09-25 | Micrology Laboratories, Llc | Test media for quantitative or qualitative identification and differentiation of general coliforms, E coli, Aeromonas spp and Salmonella spp materials in a test sample |
| WO2001025476A1 (en) * | 1999-10-01 | 2001-04-12 | Applied Imaging Corporation | Simultaneous enzymatic detection of two analytes |
| FR2810676B1 (en) * | 2000-06-27 | 2003-03-07 | Alain Rambach | CULTURE MEDIUM FOR THE DETECTION AND / OR DISCRIMINATION OF VIBRIO BACTERIA AND METHOD OF IMPLEMENTING |
| WO2002033396A1 (en) | 2000-10-14 | 2002-04-25 | Saunders Alex M | Ligand based solution assay for low concentration analytes |
| FR2816955B1 (en) * | 2000-11-17 | 2004-11-26 | Biomerieux Sa | SUBSTRATE, COMBINED SUBSTRATE, CULTURE MEDIUM AND METHOD FOR IDENTIFYING LISTERIA MONOCYTOGENES |
| FR2816956B1 (en) | 2000-11-17 | 2004-08-20 | Bio Merieux | METHOD AND MEDIUM FOR DETECTION / IDENTIFICATION OF BACTERIA WITH ESTERASIC ACTIVITY |
| FR2819266B1 (en) * | 2001-01-05 | 2004-01-16 | Alain Rambach | CULTURE MEDIUM FOR THE DETECTION AND / OR DISCRIMINATION OF LISTERIA BACTERIA AND METHOD OF IMPLEMENTING |
| FR2822847B1 (en) * | 2001-03-30 | 2003-05-09 | Bio Merieux | SPECIFIC DETECTION MEDIA FOR STAPHYLOCOCCUS AUREUS AND IDENTIFICATION AND / OR ENUMERATION METHOD USING SUCH MEDIA |
| JP4886122B2 (en) * | 2001-05-31 | 2012-02-29 | 日水製薬株式会社 | Vibrio parahaemolyticus detection medium |
| FR2826019B1 (en) * | 2001-06-13 | 2003-09-26 | Alain Rambach | CULTURE MEDIUM FOR DETECTION AND / OR DISCRIMINATION OF ENTEROCOCCUS AND METHOD OF IMPLEMENTATION |
| FR2833613B1 (en) * | 2001-12-13 | 2004-08-27 | Alain Rambach | DETECTION METHOD FOR MICROORGANISMS |
| CU23302A1 (en) | 2003-01-10 | 2008-07-24 | Ct Nac Biopreparados | SELECTIVE CROP MEANS FOR THE INSULATION AND DETECTION OF GÉ0 / 00NERO STREPTOCOCCUS SPECIES |
| JP2006025608A (en) * | 2004-07-12 | 2006-02-02 | Chisso Corp | Microorganism culture medium |
| FR2875241B1 (en) * | 2004-09-16 | 2010-07-30 | Biomerieux Sa | METHOD OF DETECTING STREPTOCOCCUS AGALACTIAE USING ESTERASE ACTIVITY |
| FR2881755B1 (en) | 2005-02-10 | 2012-11-30 | Biomerieux Sa | MEDIA FOR THE SPECIFIC DETECTION OF RESISTANT MICROORGANISMS |
| FR2882370B1 (en) * | 2005-02-22 | 2010-12-03 | Alain Rambach | DETECTION OF A MICROORGANISM STRAIN IN A LIQUID SAMPLE |
| US7749724B2 (en) * | 2005-07-05 | 2010-07-06 | Washington State University | Fluorogenic selective and differential medium for isolation of Enterobacter sakazakii |
| FR2897874B1 (en) * | 2006-02-28 | 2013-11-15 | Biomerieux Sa | METHOD FOR THE IDENTIFICATION OF AT LEAST TWO GROUPS OF MICROORGANISMS |
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| US5464755A (en) * | 1994-04-29 | 1995-11-07 | Biolog, Inc. | Microbiological medium and method of assay |
| US5510243A (en) * | 1994-06-21 | 1996-04-23 | Gelman Sciences, Inc. | Multiple chromogen enzyme targeting (MCET) for use in bacterial contamination monitoring |
-
1993
- 1993-07-28 FR FR9309294A patent/FR2708286B1/en not_active Expired - Fee Related
-
1994
- 1994-07-28 US US08/591,611 patent/US5962251A/en not_active Expired - Lifetime
- 1994-07-28 JP JP50562495A patent/JP3407302B2/en not_active Expired - Lifetime
- 1994-07-28 ES ES94923761T patent/ES2114697T3/en not_active Expired - Lifetime
- 1994-07-28 EP EP94923761A patent/EP0711360B1/en not_active Expired - Lifetime
- 1994-07-28 AT AT94923761T patent/ATE163972T1/en active
- 1994-07-28 DE DE69408993T patent/DE69408993T2/en not_active Expired - Lifetime
- 1994-07-28 DK DK94923761T patent/DK0711360T3/en active
- 1994-07-28 CA CA002168114A patent/CA2168114C/en not_active Expired - Lifetime
- 1994-07-28 WO PCT/FR1994/000958 patent/WO1995004157A1/en active IP Right Grant
Also Published As
| Publication number | Publication date |
|---|---|
| US5962251A (en) | 1999-10-05 |
| ES2114697T3 (en) | 1998-06-01 |
| EP0711360A1 (en) | 1996-05-15 |
| FR2708286B1 (en) | 1995-10-20 |
| CA2168114A1 (en) | 1995-02-09 |
| CA2168114C (en) | 2004-12-14 |
| JPH09500791A (en) | 1997-01-28 |
| EP0711360B1 (en) | 1998-03-11 |
| ATE163972T1 (en) | 1998-03-15 |
| DK0711360T3 (en) | 1998-12-21 |
| DE69408993T2 (en) | 1998-10-01 |
| WO1995004157A1 (en) | 1995-02-09 |
| DE69408993D1 (en) | 1998-04-16 |
| FR2708286A1 (en) | 1995-02-03 |
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